Lettuce variety red bluff

ABSTRACT

A lettuce cultivar, designated Red Bluff, is disclosed. The invention relates to the seeds and plants of lettuce cultivar Red Bluff and to methods for producing a lettuce plant by crossing the cultivar Red Bluff with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants and plant parts produced by those methods. This invention also relates to lettuce cultivars or breeding cultivars and plant parts derived from lettuce cultivar Red Bluff, to methods for producing other lettuce cultivars, lines or plant parts derived from lettuce cultivar Red Bluff and to the lettuce plants, varieties, and their parts derived from the use of those methods. The invention further relates to hybrid lettuce seeds, plants, and plant parts produced by crossing cultivar Red Bluff with another lettuce cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a red leaf lettuce (Lactuca sativa L.)variety designated Red Bluff. All publications cited in this applicationare herein incorporated by reference.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Lactuca sativa that is grown for its ediblehead and leaves. Lactuca sativa is in the Cichoreae tribe of theAsteraceae (Compositae) family. Lettuce is related to chicory,sunflower, aster, dandelion, artichoke, and chrysanthemum. Sativa is oneof about 300 species in the genus Lactuca. There are seven differentmorphological types of lettuce. The crisphead group includes the icebergand batavian types. Iceberg lettuce has a large, firm head with a crisptexture and a white or creamy yellow interior. The batavian lettucepredates the iceberg type and has a smaller and less firm head. Thebutterhead group has a small, soft head with an almost oily texture. Theromaine, also known as cos lettuce, has elongated upright leaves forminga loose, loaf-shaped head and the outer leaves are usually dark green.Leaf lettuce comes in many varieties, none of which form a head, andinclude the green oak leaf variety. Latin lettuce looks like a crossbetween romaine and butterhead. Stem lettuce has long, narrow leaves andthick, edible stems. Oilseed lettuce is a type grown for its large seedsthat are pressed to obtain oil. Latin lettuce, stem lettuce, and oilseedlettuce are seldom seen in the United States.

There is an ongoing need for improved lettuce varieties. Presently,there are over a thousand known lettuce cultivars. As a crop, lettuce isgrown commercially wherever environmental conditions permit theproduction of an economically viable yield. Lettuce is the World's mostpopular salad.

The goal of lettuce plant breeding is to develop new, unique, andsuperior lettuce cultivars. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing, and mutations. The breeder has no direct control at thecellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same lettuce traits. Eachyear, the plant breeder selects the germplasm to advance to the nextgeneration. This germplasm is grown under different geographical,climatic, and soil conditions, and further selections are then madeduring, and at the end of, the growing season. The cultivars that aredeveloped are unpredictable. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very gross and general fashion. The same breeder cannot produce thesame line twice by using the exact same original parents and the sameselection techniques.

The development of commercial lettuce cultivars requires the developmentof lettuce varieties, the crossing of these varieties, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding, John Wiley and Son, pp.115-161 (1960); Allard (1960); Simmonds (1979); Sneep, et al. (1979);Fehr (1987); “Carrots and Related Vegetable Umbelliferae,” Rubatzky, V.E., et al. (1999).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts, as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Lettuce in general, and leaf lettuce in particular, is an important andvaluable vegetable crop. Thus, a continuing goal of lettuce plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. To accomplish this goal, the lettuce breeder mustselect and develop lettuce plants with traits that result in superiorcultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel lettuce cultivardesignated Red Bluff. This invention thus relates to the seeds oflettuce cultivar Red Bluff, to the plants of lettuce cultivar Red Bluff,and to methods for producing a lettuce plant produced by crossing thelettuce cultivar Red Bluff with itself or another lettuce plant, tomethods for producing a lettuce plant containing in its genetic materialone or more transgenes, and to the transgenic lettuce plants produced bythat method. This invention also relates to methods for producing otherlettuce cultivars derived from lettuce cultivar Red Bluff and to thelettuce cultivar derived by the use of those methods. This inventionfurther relates to hybrid lettuce seeds and plants produced by crossinglettuce cultivar Red Bluff with another lettuce variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar Red Bluff. The tissue culturewill preferably be capable of regenerating plants having essentially allof the physiological and morphological characteristics of the foregoinglettuce plant, and of regenerating plants having substantially the samegenotype as the foregoing lettuce plant. Preferably, the regenerablecells in such tissue cultures will be callus, protoplasts, meristematiccells, cotyledons, hypocotyl, leaves, pollen, embryos, roots, root tips,anthers, pistils, shoots, stems, petiole flowers, and seeds. Stillfurther, the present invention provides lettuce plants regenerated fromthe tissue cultures of the invention.

Another aspect of the invention is to provide methods for producingother lettuce plants derived from lettuce cultivar Red Bluff. Lettucecultivars derived by the use of those methods are also part of theinvention.

The invention also relates to methods for producing a lettuce plantcontaining in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods.

In another aspect, the present invention provides for single geneconverted plants of Red Bluff. The single transferred gene maypreferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as male sterility, herbicideresistance, insect or pest resistance, modified fatty acid metabolism,modified carbohydrate metabolism, resistance for bacterial, fungal, orviral disease, male fertility, enhanced nutritional quality, andindustrial usage. The single gene may be a naturally occurring lettucegene or a transgene introduced through genetic engineering techniques.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection, and transformation. Seeds, lettuce plants,and parts thereof, produced by such breeding methods are also part ofthe invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Big Vein virus. Big vein is a disease of lettuce caused by LettuceMirafiori Big Vein Virus which is transmitted by the fungus Olpidiumvirulentus, with vein clearing and leaf shrinkage resulting in plants ofpoor quality and reduced marketable value.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants.

Bremia lactucae. An Oomycete that causes downy mildew in lettuce incooler growing regions.

Core length. Length of the internal lettuce stem measured from the baseof the cut and trimmed head to the tip of the stem.

Corky root. A disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

Cotyledon. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem.

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Lettuce Mosaic virus. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Maturity date. Maturity refers to the stage when the plants are of fullsize or optimum weight, in marketable form or shape to be of commercialor economic value.

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter. Head height divided by the head diameteris an indication of the head shape; <1 is flattened, 1=round, and >1 ispointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing or via genetic engineering wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe backcrossing technique or via genetic engineering.

Tip burn. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

Tomato Bushy Stunt. A disease which causes stunting of growth, leafmottling, and deformed or absent fruit.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce Red bluff is a red leaf lettuce variety suitable for full sizeproduction in the coastal areas of California in the Spring and Fallharvesting seasons, and the southwest deserts of California and Arizonain the winter harvesting season. Lettuce variety Red Bluff resulted froma cross of a red leaf lettuce variety with a DMR lettuce line with DMgene R37 released from UC Davis and subsequent numerous generations ofindividual plant selections chosen for their red color and DMresistance.

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in cultivar Red Bluff.

Lettuce cultivar Red Bluff has the following morphologic and othercharacteristics, described in Table 1.

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant: Type: Red Leaf Maturitydate: 65 days (Summer), 85 days (winter) from first water date. Seed:Color: Black Light dormancy: Light not required Heat dormancy:Susceptible Cotyledon: Shape: Broad Fourth leaf: Apical Margin: EntireBasal Margin: Entire Undulation: Flat Anthocyanin distribution:Throughout Anthocyanin Concentration: Moderate Rolling: Absent Cupping:Uncupped Reflexing: None Mature Leaves: Margin: Incision depth:Absent/Shallow Indentation: Shallowly Dentate Undulation of the apicalmargin: Moderate Anthocyanin distribution: Throughout AnthocyaninConcentration: Moderate Glossiness: Moderate Blistering: Absent/SlightThickness: Intermediate Trichomes: Absent Plant at Market Stage Headshape: Non-heading Head size class: Medium Head weight (g): 503.2 Headfirmness: loose Core: Diameter at base of head (mm): 32.6 Core heightfrom base of head to apex (mm): 57.2 Primary Regions of Adaptation:Spring area: Salinas, Imperial, California, and Yuma, Arizona (UnitedStates) Autumn area: Salinas, California (United States) Winter area:Yuma, Arizona, Imperial and Coachella, California (United States)Disease and Stress Reactions: Downy Mildew (Bremia lactucae): Highlyresistant Big Vein: Intermediate Tipburn: Susceptable Heat: IntermediateCold: Tolerant Brown Rib: Resistant Pink Rib: Resistant Rusty BrownDiscoloration: Resistant Internal Rib Necrosis (Blackheart, Gray Rib,Gray Streak): Resistant

FURTHER EMBODIMENTS OF THE INVENTION

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Any DNA sequences,whether from a different species or from the same species, which areintroduced into the genome using transformation or various breedingmethods, are referred to herein collectively as “transgenes.” Over thelast fifteen to twenty years, several methods for producing transgenicplants have been developed, and the present invention, in particularembodiments, also relates to transformed versions of the claimed line.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine,and others can also be used for antisense, dsRNA, and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the invention may beproduced by any means, including genomic preparations, cDNApreparations, in vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedlettuce plants using transformation methods as described below toincorporate transgenes into the genetic material of the lettuceplant(s).

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformed plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley, et al., PNAS, 80:4803 (1983).Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford, et al., Plant Physiol.,86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, etal., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol.,7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil. Comai, etal., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell,2:603-618 (1990); and Stalker, et al., Science, 242:419-423 (1988).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase, and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol., 5:387 (1987); Teeri, et al., EMBOJ., 8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); and DeBlock, etal., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151 a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter, therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Melt, et al., PNAS, 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991) and Gatz, et al.,Mol. Gen. Genet., 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, etal., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena,et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression inlettuce or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, Xba1/Ncol fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xba1/Ncol fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expressionin lettuce. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in lettuce. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is lettuce. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., 269:284, CRC Press, Boca Raton(1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

A. Genes that Confer Resistance to Pests or Disease and that Encode

1. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science, 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);and Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995, and31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

4. A vitamin-binding protein such as avidin. See PCT Application No. US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus α-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient released by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb, et al., Bio/technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J, 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

19. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant, et al., Mol. Breeding, 3:1, 75-86 (1997).

Any of the above listed disease or pest resistance genes (1-19) can beintroduced into the claimed lettuce cultivar through a variety of meansincluding but not limited to transformation and crossing.

B. Genes that Confer Resistance to an Herbicide:

Exemplary polynucleotides encoding polypeptides that confer traitsdesirable for herbicide resistance include acetolactate synthase (ALS)mutants that lead to herbicide resistance such as the S4 and/or Hramutations ((resistance to herbicides including sulfonylureas,imidazolinones, triazolopyrimidines, pyrimidinyl thiobenzoates);glyphosate resistance (e.g.,5-enol-pyrovyl-shikimate-3-phosphate-synthase (EPSPS) gene, includingbut not limited to those described in U.S. Pat. Nos. 4,940,935,5,188,642, 5,633,435, 6,566,587, 7,674,598 as well as all relatedapplication; or the glyphosate N-acetyltransferase (GAT) gene, describedin Castle et al., Science, 2004, 304:1151-1154; and in U.S. PatentApplication Publication Nos. 20070004912, 20050246798, and20050060767)); glufosinate resistance (e.g, BAR; see e.g., U.S. Pat. No.5,561,236); 2,4-D resistance (e.g. aryloxy alkanoate dioxygenase orAAD-1, AAD-12, or AAD-13), HPPD resistance (e.g. Pseudomonas HPPD) andPPO resistance (e.g., fomesafen, acifluorfen-sodium, oxyfluorfen,lactofen, fluthiacet-methyl, saflufenacil, flumioxazin,flumiclorac-pentyl, carfentrazone-ethyl, sulfentrazone,); a cytochromeP450 or variant thereof that confers herbicide resistance or toleranceto, inter alia, HPPD-inhibitingherbicides, PPO-inhibiting herbicides andALS-inhibiting herbicides (U.S. Patent Application Publication No.20090011936; U.S. Pat. Nos. 6,380,465; 6,121,512; 5,349,127; 6,649,814;and 6,300,544; and PCT International Publication No. WO 2007/000077);dicamba resistance (e.g. dicamba monoxygenase), and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; PCT International Publication No. WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE), and starch debranching enzymes (SDBE));and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al., J. Bacteriol., 1988, 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosuresof which are herein incorporated by reference.

Any of the above listed herbicide genes can be introduced into theclaimed lettuce cultivar through a variety of means including, but notlimited to, transformation and crossing.

C. Genes that Confer or Contribute to a Value-Added Trait, Such as:

1. Increased iron content of the lettuce, for example, by introducinginto a plant a soybean ferritin gene as described in Goto, et al., ActaHorticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa lettuce a gene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the lettuce by introducing a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia, et al., Bio/technology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into aplant an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon, et al., PNAS, 89:2625 (1992).

5. Modified carbohydrate composition effected, for example, byintroducing into plants a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteriol., 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene);Pen, et al., Bio/technology, 10:292 (1992) (production of transgenicplants that express Bacillus lichenifornnis α-amylase); Elliot, et al.,Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomatoinvertase genes); Søgaard, et al., J. Biol. Chem., 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher, etal., Plant Physiol., 102:1045 (1993) (maize endosperm starch branchingenzyme II).

D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT. See International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See InternationalPublications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Torres, et al., PlantCell Tissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al.,Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep., 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer:

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 μm to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, January),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2,October), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12(9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150(1996); Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996);Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C.,Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563(1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al.,Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J,4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptakeof DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.,199:161 (1985) and Draper, et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M., Kuhne, T., Biologia Plantarum, 40(4):507-514(1997/98); Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, etal., Plant Cell, 4:1495-1505 (1992); and Spencer, et al., Plant Mol.Biol., 24:51-61 (1994). See also Chupean, et al., Bio/technology, 7:5,503-508 (1989).

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossedwith another (non-transformed or transformed) line in order to produce anew transgenic lettuce line. Alternatively, a genetic trait which hasbeen engineered into a particular lettuce cultivar using the foregoingtransformation techniques could be introduced into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Gene Conversions

When the term “lettuce plant” is used in the context of the presentinvention, this also includes any gene conversions of that variety. Theterm “gene converted plant” as used herein refers to those lettuceplants which are developed by backcrossing, genetic engineering, ormutation, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more genes transferred into the variety via the backcrossingtechnique, genetic engineering, or mutation. Backcrossing methods can beused with the present invention to improve or introduce a characteristicinto the variety. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to the recurrent parent,i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to therecurrent parent. The parental lettuce plant which contributes the genefor the desired characteristic is termed the “nonrecurrent” or “donorparent.” This terminology refers to the fact that the nonrecurrentparent is used one time in the backcross protocol and therefore does notrecur. The parental lettuce plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol. Poehlman &Sleper (1994) and Fehr (1993). In a typical backcross protocol, theoriginal variety of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until alettuce plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the transferredgene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the original line.To accomplish this, a gene of the recurrent cultivar is modified orsubstituted with the desired gene from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired physiological and morphological, constitution ofthe original line. The choice of the particular nonrecurrent parent willdepend on the purpose of the backcross. One of the major purposes is toadd some commercially desirable, agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selected inthe development of a new line but that can be improved by backcrossingtechniques. Gene traits may or may not be transgenic. Examples of thesetraits include, but are not limited to, male sterility, modified fattyacid metabolism, modified carbohydrate metabolism, herbicide resistance,resistance for bacterial, fungal, or viral disease, insect resistance,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Several of these gene traits are described in U.S. Pat. Nos.5,777,196, 5,948,957, and 5,969,212, the disclosures of which arespecifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants having thephysiological and morphological characteristics of variety Red Bluff.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a lettuceplant of cultivar Red Bluff. Further, both first and second parentlettuce plants can come from lettuce cultivar Red Bluff. Thus, any suchmethods using lettuce cultivar Red Bluff are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using lettuce cultivar Red Bluff as at leastone parent are within the scope of this invention, including thosedeveloped from cultivars derived from lettuce cultivar Red Bluff.Advantageously, this lettuce cultivar could be used in crosses withother, different, lettuce plants to produce the first generation (F₁)lettuce hybrid seeds and plants with superior characteristics. Thecultivar of the invention can also be used for transformation whereexogenous genes are introduced and expressed by the cultivar of theinvention. Genetic variants created either through traditional breedingmethods using lettuce cultivar Red Bluff or through transformation ofcultivar Red Bluff by any of a number of protocols known to those ofskill in the art are intended to be within the scope of this invention.

The following describes breeding methods that may be used with lettucecultivar Red Bluff in the development of further lettuce plants. Onesuch embodiment is a method for developing cultivar Red Bluff progenylettuce plants in a lettuce plant breeding program comprising: obtainingthe lettuce plant, or a part thereof, of cultivar Red Bluff, utilizingsaid plant or plant part as a source of breeding material, and selectinga lettuce cultivar Red Bluff progeny plant with molecular markers incommon with cultivar Red Bluff and/or with morphological and/orphysiological characteristics selected from the characteristics listedin Table 1. Breeding steps that may be used in the lettuce plantbreeding program include pedigree breeding, backcrossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers), and the making of double haploidsmay be utilized.

Another method involves producing a population of lettuce cultivar RedBluff progeny lettuce plants, comprising crossing cultivar Red Bluffwith another lettuce plant, thereby producing a population of lettuceplants, which, on average, derive 50% of their alleles from lettucecultivar Red Bluff. A plant of this population may be selected andrepeatedly selfed or sibbed with a lettuce cultivar resulting from thesesuccessive filial generations. One embodiment of this invention is thelettuce cultivar produced by this method and that has obtained at least50% of its alleles from lettuce cultivar Red Bluff.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes lettucecultivar Red Bluff progeny lettuce plants comprising a combination of atleast two cultivar Red Bluff traits selected from the group consistingof those listed in Table 1 or the cultivar Red Bluff combination oftraits listed in the Summary of the Invention, so that said progenylettuce plant is not significantly different for said traits thanlettuce cultivar Red Bluff as determined at the 5% significance levelwhen grown in the same environmental conditions. Using techniquesdescribed herein, molecular markers may be used to identify said progenyplant as a lettuce cultivar Red Bluff progeny plant. Mean trait valuesmay be used to determine whether trait differences are significant, andpreferably the traits are measured on plants grown under the sameenvironmental conditions. Once such a variety is developed, its value issubstantial since it is important to advance the germplasm base as awhole in order to maintain or improve traits such as yield, diseaseresistance, pest resistance, and plant performance in extremeenvironmental conditions.

Progeny of lettuce cultivar Red Bluff may also be characterized throughtheir filial relationship with lettuce cultivar Red Bluff, as forexample, being within a certain number of breeding crosses of lettucecultivar Red Bluff. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between lettuce cultivar Red Bluff and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4, or 5 breeding crosses of lettuce cultivar Red Bluff.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, stems, and the like.

TABLES

Table 2 compares the length of the cotyledon leaf in millimeters of 20day old seedlings of lettuce cultivar Red Bluff with commercial lettucecultivars Red Tide and Red Rage and shows the ANOVA results thatindicate a significant difference in the cotyledon length between thevarieties at 20 days old. Data were taken in 2011 in Gilroy, Calif. on20 plants of each variety.

TABLE 2 Cotyledon length (mm) Red Bluff Red Tide Red Rage 12 16 10 11 1315 12 14 15 11 15 16 12 14 12 13 13 7 12 14 10 10 11 9 13 19 13 13 14 1210 15 10 13 10 12 10 16 15 13 13 15 10 8 10 10 15 10 9 13 12 11 14 13 1316 13 11 15 10 Anova: Single Factor SUMMARY Groups Count Sum AverageVariance Red Bluff 20 229 11.45 1.734211 Red Tide 20 278 13.9 5.568421Red Rage 20 239 11.95 5.944737 ANOVA Source of Variation SS df MS FP-value F crit Between 67.033333 2 33.51667 7.590187 0.001195 3.158846Groups Within 251.7 57 4.415789 Groups Total 318.73333 59Table 3 compares the width of the cotyledon leaf in millimeters of 20day old seedlings of lettuce cultivar Red Bluff with commercial lettucecultivars Red Tide and Red Rage and shows the ANOVA results thatindicate a significant difference in the cotyledon width between thevarieties at 20 days old. Data were taken in 2011 in Gilroy, Calif. on20 plants of each variety.

TABLE 3 Cotyledon Width (mm) Red Bluff Red Tide Red Rage 8 11 6 7 8 10 810 9 7 10 8 9 9 7 7 10 6 7 10 5 7 7 6 7 11 8 7 9 7 8 10 5 8 6 6 7 10 6 79 9 6 7 6 7 9 7 9 8 7 8 10 10 7 8 9 8 10 7 Anova: Single Factor SUMMARYGroups Count Sum Average Variance Red Bluff 20 149 7.45 0.576316 RedTide 20 182 9.1 1.884211 Red Rage 20 144 7.2 2.378947 ANOVA Source ofVariation SS df MS F P-value F crit Between 42.633333 2 21.3166713.21425 1.93E−05 3.158846 Groups Within 91.95 57 1.613158 Groups Total134.58333 59Table 4 compares the cotyledon leaf index of 20 day old seedlings oflettuce cultivar Red Bluff with commercial lettuce cultivars Red Tideand Red Rage and shows the ANOVA results that indicate a non significantdifference in the cotyledon index between the varieties at 20 days old.Data were taken in 2011 in Gilroy, Calif. on 20 plants of each variety.

TABLE 4 Cotyledon Index (calculated by dividing the cotyledon leaflength by the cotyledon leaf width) Red Bluff Red Tide Red Rage 1.5 1.51.7 1.6 1.6 1.5 1.5 1.4 1.7 1.6 1.5 2.0 1.3 1.6 1.7 1.9 1.3 1.2 1.7 1.42.0 1.4 1.6 1.5 1.9 1.7 1.6 1.9 1.6 1.7 1.3 1.5 2.0 1.6 1.7 2.0 1.4 1.62.5 1.9 1.4 1.7 1.7 1.1 1.7 1.4 1.7 1.4 1.0 1.6 1.7 1.4 1.4 1.3 1.9 2.01.4 1.4 1.5 1.4 Anova: Single Factor SUMMARY Groups Count Sum AverageVariance Red Bluff 20 31.053571 1.552679 0.056243 Red Tide 20 30.6349931.53175 0.031172 Red Rage 20 33.702778 1.685139 0.091756 ANOVA Source ofVariation SS df MS F P-value F crit Between 0.2767468 2 0.138373 2.316890.107795 3.158846 Groups Within 3.4042543 57 0.059724 Groups Total3.6810011 59Table 5 compares the length of the 4th true leaf measured in centimetersof 20 day old seedlings of lettuce cultivar Red Bluff with commerciallettuce cultivars Red Tide and Red Rage and shows the ANOVA results thatindicate significant differences in the 4th leaf length between thevarieties at 20 days old. Data were taken in 2011 in Gilroy, Calif. on20 plants of each variety.

TABLE 5 4th Leaf Length (cm) Red Bluff Red Tide Red Rage 10.7 13.9 10.613.6 7.4 11.1 13.2 8.7 12.5 13.4 9.4 9.4 7.6 9.9 10.3 14.5 10.4 5.4 119.5 7.7 11.8 10.6 11.2 12.5 14.1 13.3 12.7 12.4 11.2 5.9 8.1 11.9 12.65.1 9.8 13 6.5 11 12.6 4.7 10.7 12.4 7.1 6.3 12.4 7.8 11.8 9.5 9.5 10.413 1.5 13.5 9.9 12.5 10.5 14.3 11.4 11.9 Anova: Single Factor SUMMARYGroups Count Sum Average Variance Red Bluff 20 236.6 11.83 4.771684 RedTide 20 180.5 9.025 10.01461 Red Rage 20 210.5 10.525 4.279868 ANOVASource of Variation SS df MS F P-value F crit Between 78.807 2 39.40356.200017 0.003662 3.158846 Groups Within 362.257 57 6.355386 GroupsTotal 441.064 59Table 6 compares the width of the 4th true leaf measured in millimetersof 20 day old seedlings of leaf lettuce cultivar Red Bluff withcommercial lettuce cultivars Red Tide and Red Rage and shows the ANOVAresults that indicate significant differences in the 4th leaf widthbetween the varieties at 20 days old. Data were taken in 2011 in Gilroy,Calif. on 20 plants of each variety.

TABLE 6 4th Leaf Width (cm) Red Bluff Red Tide Red Rage 3.5 5.5 4.2 5.12.6 5 4.7 3.5 5.7 5.1 4.5 4.7 2.5 4.8 3.9 5.3 4.8 1.9 4.4 4.3 3.6 3.63.8 5.3 4.2 5.9 6.2 3.9 4.6 4.6 2.5 3.4 6.1 5.1 1.9 4.9 4.8 2.7 5 4.61.8 4.8 4.3 2.7 2.9 4.1 3.5 5.1 3.6 2.9 4.6 5.4 4.1 6.5 3.9 4.5 4.7 5.24.9 5.2 Anova: Single Factor SUMMARY Groups Count Sum Average VarianceRed Bluff 20 85.8 4.29 0.732526 Red Tide 20 76.7 3.835 1.319237 Red Rage20 94.9 4.745 1.189974 ANOVA Source of Variation SS df MS F P-value Fcrit Between 8.281 2 4.1405 3.831742 0.027457 3.158846 Groups Within61.593 57 1.080579 Groups Total 69.874 59Table 7 compares the 4th leaf index (calculated by dividing the 4th leaflength by the 4th leaf width) of 20 day old seedlings of lettucecultivar Red Bluff with commercial lettuce cultivars Red Tide and RedRage and shows the ANOVA results that indicate significant differencesin the 4th leaf index between the varieties at 20 days old. Data weretaken in 2011 in Gilroy, Calif. on 20 plants of each variety.

TABLE 7 4th Leaf Index calculated by dividing the 4th leaf length by the4th leaf width Red Bluff Red Tide Red Rage 3.1 2.5 2.5 2.7 2.8 2.2 2.82.5 2.2 2.6 2.1 2.0 3.0 2.1 2.6 2.7 2.2 2.8 2.5 2.2 2.1 3.3 2.8 2.1 3.02.4 2.1 3.3 2.7 2.4 2.4 2.4 2.0 2.5 2.7 2.0 2.7 2.4 2.2 2.7 2.6 2.2 2.92.6 2.2 3.0 2.2 2.3 2.6 3.3 2.3 2.4 0.4 2.1 2.5 2.8 2.2 2.8 2.3 2.3Anova: Single Factor SUMMARY Groups Count Sum Average Variance Red Bluff20 55.46692 2.773346 0.070602 Red Tide 20 47.950762 2.397538 0.317053Red Rage 20 44.978386 2.248919 0.048322 ANOVA Source of Variation SS dfMS F P-value F crit Between 2.9222832 2 1.461142 10.05424 0.0001823.158846 Groups Within 8.2835734 57 0.145326 Groups Total 11.205857 59Table 8 compares the plant weight (g) of lettuce cultivar Red Bluff withcommercial lettuce cultivars Red Tide and Red Rage and shows the ANOVAresults that indicate significant differences in the plant weightbetween the varieties at harvest maturity. Data were taken in 2010 and2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20 plants of eachvariety. (Raw data not shown)

TABLE 8 Plant Weight (g) at Harvest Maturity Anova: Two-Factor WithReplication SUMMARY Trial 1 Trial 2 Total Red Bluff Count 20 20 40 Sum9862 10265 20127 Average 493.1 513.25 503.175 Variance 2467.98952305.9868 2429.892 Red Tide Count 20 20 40 Sum 10090 10575 20665 Average504.5 528.75 516.625 Variance 1362.8947 769.03947 1189.42 Red Rage Count20 20 40 Sum 8256 8305 16561 Average 412.8 415.25 414.025 Variance1094.6947 1361.7763 1198.281 Total Count 60 60 Sum 28208 29145 Average470.13333 485.75 Variance 3279.6429 3996.7669 ANOVA Source of VariationSS df MS F P-value F crit Sample 248738.47 2 124369.2 79.70359 2.22E−223.075854 Columns 7316.4083 1 7316.408 4.688812 0.032443 3.924328Interaction 2684.4667 2 1342.233 0.860187 0.42581 3.075854 Within177885.25 114 1560.397 Total 436624.59 119Table 9 compares the plant height (cm) of lettuce cultivar Red Bluffwith commercial lettuce cultivars Red Tide and Red Rage and shows theANOVA results that indicate significant non differences in the plantheight between the varieties at harvest maturity. Data were taken in2010 and 2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20 plantsof each variety. (Raw data not shown)

TABLE 9 Plant Height (cm) at Harvest Maturity Anova: Two-Factor WithReplication SUMMARY Trial 1 Trial 2 Total Red Bluff Count 20 20 40 Sum522 546 1068 Average 26.1 27.3 26.7 Variance 2.3052632 1.90526322.420513 Red Tide Count 20 20 40 Sum 528 520 1048 Average 26.4 26 26.2Variance 2.0421053 1.1578947 1.6 Red Rage Count 20 20 40 Sum 513 5271040 Average 25.65 26.35 26 Variance 2.0289474 1.8184211 2 Total Count60 60 Sum 1563 1593 Average 26.05 26.55 Variance 2.15 1.8788136 ANOVASource of Variation SS df MS F P-value F crit Sample 10.4 2 5.2 2.7713880.066795 3.075854 Columns 7.5 1 7.5 3.997195 0.047955 3.924328Interaction 13.4 2 6.7 3.570827 0.031322 3.075854 Within 213.9 1141.876316 Total 245.2 119Table 10 compares the frame leaf length (cm) of lettuce cultivar RedBluff with commercial lettuce cultivars Red Tide and Red Rage and showsthe ANOVA results that indicate non significant differences in frameleaf length between the varieties at harvest maturity. Data were takenin 2010 and 2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20plants of each variety. (Raw data not shown)

TABLE 10 Frame Leaf Length (cm) at Harvest Maturity Anova: Two-FactorWith Replication SUMMARY Trial 1 Trial 2 Total Red Bluff Count 20 20 40Sum 504 513 1017 Average 25.2 25.65 25.425 Variance 2.2736842 1.08157891.686538 Red Tide Count 20 20 40 Sum 502 501 1003 Average 25.1 25.0525.075 Variance 1.9894737 1.2078947 1.558333 Red Rage Count 20 20 40 Sum491 505 996 Average 24.55 25.25 24.9 Variance 2.7868421 1.98684212.451282 Total Count 60 60 Sum 1497 1519 Average 24.95 25.316667Variance 2.3533898 1.4403955 ANOVA Source of Variation SS df MS FP-value F crit Sample 5.7166667 2 2.858333 1.514173 0.224381 3.075854Columns 4.0333333 1 4.033333 2.136617 0.14657 3.924328 Interaction2.9166667 2 1.458333 0.772537 0.464242 3.075854 Within 215.2 1141.887719 Total 227.86667 119Table 11 compares the frame leaf width (cm) of lettuce cultivar RedBluff with commercial lettuce cultivars Red Tide and Red Rage and showsthe ANOVA results that indicate significant differences in frame leafwidth between the varieties at harvest maturity. Data were taken in 2010and 2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20 plants ofeach variety. (Raw data not shown)

TABLE 11 Frame Leaf Width (cm) at Harvest Maturity Anova: Two-FactorWith Replication SUMMARY Trial 1 Trial 2 Total Red Bluff Count 20 20 40Sum 446 455 901 Average 22.3 22.75 22.525 Variance 1.6947368 0.82894741.28141 Red Tide Count 20 20 40 Sum 450 445 895 Average 22.5 22.2522.375 Variance 1.9473684 1.9868421 1.932692 Red Rage Count 20 20 40 Sum426 437 863 Average 21.3 21.85 21.575 Variance 2.0105263 1.60789471.840385 Total Count 60 60 Sum 1322 1337 Average 22.033333 22.283333Variance 2.100565 1.5624294 ANOVA Source of Variation SS df MS F P-valueF crit Sample 20.866667 2 10.43333 6.212588 0.002748 3.075854 Columns1.875 1 1.875 1.116479 0.292912 3.924328 Interaction 3.8 2 1.9 1.1313660.326187 3.075854 Within 191.45 114 1.679386 Total 217.99167 119Table 12 compares the frame leaf index of lettuce cultivar Red Bluffwith commercial lettuce cultivars Red Tide and Red Rage and shows theANOVA results that indicate significant differences in frame leaf indexbetween the varieties at harvest maturity. Data were taken in 2010 and2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20 plants of eachvariety. (Raw data not shown)

TABLE 12 Harvest Mature Leaf Index (calculated by dividing the leaflength by the leaf width) Anova: Two-Factor With Replication SUMMARYTrial 1 Trial 2 Total Red Bluff Count 20 20 40 Sum 22.605341 22.56305745.1684 Average 1.130267 1.1281528 1.12921 Variance 0.0008328 0.00173180.001251 Red Tide Count 20 20 40 Sum 22.331733 22.554888 44.88662Average 1.1165866 1.1277444 1.122166 Variance 0.0014442 0.00192520.001673 Red Rage Count 20 20 40 Sum 23.062355 23.125184 46.18754Average 1.1531178 1.1562592 1.154688 Variance 0.0016862 0.00131480.001465 Total Count 60 60 Sum 67.999429 68.243128 Average 1.13332381.1373855 Variance 0.0015072 0.0017822 ANOVA Source of Variation SS dfMS F P-value F crit Sample 0.0234203 2 0.01171 7.86361 0.000632 3.075854Columns 0.0004949 1 0.000495 0.332344 0.565419 3.924328 Interaction0.0008934 2 0.000447 0.299978 0.741417 3.075854 Within 0.169764 1140.001489 Total 0.1945726 119Table 13 compares the frame leaf area (cm2, calculated by multiplyingthe leaf length by the leaf width) of lettuce cultivar Red Bluff withcommercial lettuce cultivars Red Tide and Red Rage and shows the ANOVAresults that indicate significant differences in the frame leaf areabetween the varieties at harvest maturity. Data were taken in 2010 and2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20 plants of eachvariety. (Raw data not shown)

TABLE 13 Leaf Area (cm2, calculated by multiplying the leaf length bythe leaf width) Anova: Two-Factor With Replication SUMMARY Trial 1 Trial2 Total Red Bluff Count 20 20 40 Sum 11273 11681 22954 Average 563.65584.05 573.85 Variance 4044.45 1736.1553 2922.9 Red Tide Count 20 20 40Sum 11326 11171 22497 Average 566.3 558.55 562.425 Variance 4008.22113143.1026 3499.379 Red Rage Count 20 20 40 Sum 10497 11063 21560 Average524.85 553.15 539 Variance 4353.3974 3659.1868 4108.923 Total Count 6060 Sum 33096 33915 Average 551.6 565.25 Variance 4360.2102 2934.3263ANOVA Source of Variation SS df MS F P-value F crit Sample 25250.45 212625.23 3.616763 0.029997 3.075854 Columns 5589.675 1 5589.675 1.6012810.208302 3.924328 Interaction 7181.45 2 3590.725 1.028639 0.3607873.075854 Within 397945.75 114 3490.752 Total 435967.33 119Table 14 compares the core length (mm) of lettuce cultivar Red Bluffwith commercial lettuce cultivars Red Tide and Red Rage and shows theANOVA results that indicate significant differences in the core lengthbetween the varieties at harvest maturity. Data were taken in 2010 and2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20 plants of eachvariety. (Raw data not shown)

TABLE 14 Core Length (mm) at Harvest Maturity Anova: Two-Factor WithReplication SUMMARY Trial 1 Trial 2 Total Red Bluff Count 20 20 40 Sum1157 1132 2289 Average 57.85 56.6 57.225 Variance 37.713158 69.09473752.43526 Red Tide Count 20 20 40 Sum 1361 1270 2631 Average 68.05 63.565.775 Variance 25.734211 48.894737 41.66603 Red Rage Count 20 20 40 Sum1667 1667 3334 Average 83.35 83.35 83.35 Variance 21.397368 33.71315826.84872 Total Count 60 60 Sum 4185 4069 Average 69.75 67.816667Variance 139.00424 179.60989 ANOVA Source of Variation SS df MS FP-value F crit Sample 14193.317 2 7096.658 180.006 5.29E−36 3.075854Columns 112.13333 1 112.1333 2.844251 0.094436 3.924328 Interaction110.51667 2 55.25833 1.401622 0.250407 3.075854 Within 4494.4 11439.42456 Total 18910.367 119Table 15 compares the diameter length (mm) of lettuce cultivar Red Bluffwith commercial lettuce cultivars Red Tide and Red Rage and shows theANOVA results that indicate significant differences in the core diameterbetween the varieties at harvest maturity. Data were taken in 2010 and2011 in San Juan Bautista, Calif. and Yuma, Ariz. for 20 plants of eachvariety. (Raw data not shown)

TABLE 15 Core Diameter (cm) at Harvest Maturity Anova: Two-Factor WithReplication SUMMARY Trial 1 Trial 2 Total Red Bluff Count 20 20 40 Sum641 663 1304 Average 32.05 33.15 32.6 Variance 4.1552632 7.18684215.835897 Red Tide Count 20 20 40 Sum 646 647 1293 Average 32.3 32.3532.325 Variance 5.5894737 4.9763158 5.148077 Red Rage Count 20 20 40 Sum507 540 1047 Average 25.35 27 26.175 Variance 9.0815789 4 7.071154 TotalCount 60 60 Sum 1794 1850 Average 29.9 30.833333 Variance 16.6 12.785311ANOVA Source of Variation SS df MS F P-value F crit Sample 1055.7167 2527.8583 90.51722 2.89E−24 3.075854 Columns 26.133333 1 26.133334.481348 0.036441 3.924328 Interaction 13.216667 2 6.608333 1.1331980.325602 3.075854 Within 664.8 114 5.831579 Total 1759.8667 119Table 16 compares the seed stalk height measured in centimeters oflettuce cultivar Red Bluff with commercial lettuce cultivars Red Tideand Red Rage and shows the ANOVA results that indicate non significantdifferences in the seed stalk height between the varieties. Data weretaken in 2011 in Bottonwillow, Calif. on 20 plants of each variety.

TABLE 16 Seed Stalk Height (cm) Red Bluff Red Tide Red Rage 89 85 85 7588 80 76 85 70 77 75 75 90 90 85 88 90 75 79 85 85 80 75 70 75 76 75 7478 77 70 80 78 69 85 80 70 75 85 71 77 88 80 75 91 85 72 90 76 70 85 7480 86 72 85 87 71 78 90 Anova: Single Factor SUMMARY Groups Count SumAverage Variance Red Bluff 20 1541 77.05 42.47105 Red Tide 20 1604 80.236.06316 Red Rage 20 1637 81.85 42.87105 ANOVA Source of Variation SS dfMS F P-value F crit Between 237.9 2 118.95 2.939329 0.060966 3.158846Groups Within 2306.7 57 40.46842 Groups Total 2544.6 59Table 17 compares the seed stalk spread measured in centimeters oflettuce cultivar Red Bluff with commercial lettuce cultivars Red Tideand Red Rage and shows the ANOVA results that indicate non significantdifferences in the seed stalk spread between the varieties. Data weretaken in 2011 in Bottonwillow, Calif. on 20 plants of each variety.

TABLE 17 Seed Stalk Spread (cm) Red Bluff Red Tide Red Rage 50 55 45 5460 50 49 45 46 45 45 47 50 45 48 51 50 50 47 52 51 49 48 45 48 50 44 4550 40 47 48 40 44 44 42 42 46 45 47 45 44 49 50 45 50 46 46 42 42 47 4040 48 41 41 49 44 42 52 Anova: Single Factor SUMMARY Groups Count SumAverage Variance Red Bluff 20 934 46.7 13.90526 Red Tide 20 944 47.224.06316 Red Rage 20 924 46.2 11.11579 ANOVA Source of Variation SS dfMS F P-value F crit Between 10 2 5 0.305597 0.737883 3.158846 GroupsWithin 932.6 57 16.3614 Groups Total 942.6 59

DEPOSIT INFORMATION

Applicants have made a deposit of at least 2500 seeds of with theAmerican Type Culture Collection (ATCC), Manassas, Va., 20110-2209U.S.A., ATCC Deposit No: PTA-12301. This deposit of the Lettuce VarietyRed Bluff will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the effective life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.Additionally, Applicants have satisfied all the requirements of 37C.F.R. §§1.801-1.809, including providing an indication of the viabilityof the sample. Applicants impose no restrictions on the availability ofthe deposited material from the ATCC; however, Applicants have noauthority to waive any restrictions imposed by law on the transfer ofbiological material or its transportation in commerce. Applicants do notwaive any infringement of its rights granted under this patent or underthe Plant Cultivar Protection Act (7 USC 2321 et seq.).

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the American Type Culture Collection, Manassas, Va.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A seed of lettuce cultivar Red Bluff, wherein arepresentative sample of seed of said cultivar was deposited under ATCCAccession No. PTA-12301.
 2. A lettuce plant, or a part thereof, producedby growing the seed of claim
 1. 3. A tissue culture produced fromprotoplasts or cells from the plant of claim 2, wherein said cells orprotoplasts are produced from a plant part selected from the groupconsisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematiccell root, root tip, pistil, anther, ovule, flower, shoot, stem, seed,and petiole.
 4. A lettuce plant regenerated from the tissue culture ofclaim 3, wherein the plant has all of the morphological andphysiological characteristics of cultivar Red Bluff.
 5. A method forproducing a lettuce seed comprising crossing two lettuce plants andharvesting the resultant lettuce seed, wherein at least one lettuceplant is the lettuce plant of claim
 2. 6. A lettuce seed produced by themethod of claim
 5. 7. A lettuce plant, or a part thereof, produced bygrowing said seed of claim
 6. 8. The method of claim 5, wherein at leastone of said lettuce plants is transgenic.
 9. A method of producing amale sterile lettuce plant, wherein the method comprises introducing anucleic acid molecule that confers male sterility into the lettuce plantof claim
 2. 10. A male sterile lettuce plant produced by the method ofclaim
 9. 11. A method of producing an herbicide resistant lettuce plant,wherein said method comprises introducing a gene conferring herbicideresistance into the plant of claim 2, wherein the gene confersresistance to an herbicide selected from the group consisting ofglyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxyproprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione,triazine, and benzonitrile.
 12. An herbicide resistant lettuce plantproduced by the method of claim
 11. 13. A method of producing a pest orinsect resistant lettuce plant, wherein said method comprisesintroducing a gene conferring pest or insect resistance into the plantof claim
 2. 14. A pest or insect resistant lettuce plant produced by themethod of claim
 13. 15. The lettuce plant of claim 14, wherein the geneencodes a Bacillus thuringiensis endotoxin.
 16. A method of producing adisease resistant lettuce plant, wherein said method comprisesintroducing a gene conferring disease resistance into the plant of claim2.
 17. A disease resistant lettuce plant produced by the method of claim16.
 18. A method of producing a lettuce plant with a value-added trait,wherein said method comprises introducing a gene conferring avalue-added trait into the plant of claim 2, where said gene encodes aprotein selected from the group consisting of a ferritin, a nitratereductase, and a monellin.
 19. A lettuce plant with a value-added traitproduced by the method of claim
 18. 20. A method of introducing adesired trait into lettuce cultivar Red Bluff wherein the methodcomprises: (a) crossing a Red Bluff plant, wherein a representativesample of seed was deposited under ATCC Accession No. PTA-12301, with aplant of another lettuce cultivar that comprises a desired trait toproduce progeny plants wherein the desired trait is selected from thegroup consisting of male sterility, herbicide resistance, insect or pestresistance, modified bolting and resistance to bacterial disease, fungaldisease or viral disease; (b) selecting one or more progeny plants thathave the desired trait to produce selected progeny plants; (c) crossingthe selected progeny plants with the Red Bluff plant to producebackcross progeny plants; (d) selecting for backcross progeny plantsthat have the desired trait and all of the physiological andmorphological characteristics of lettuce cultivar Red Bluff listed inTable 1; and (e) repeating steps (c) and (d) two or more times insuccession to produce selected third or higher backcross progeny plantsthat comprise the desired trait and all of the physiological andmorphological characteristics of lettuce cultivar Red Bluff listed inTable
 1. 21. A lettuce plant produced by the method of claim 20, whereinthe plant has the desired trait.
 22. The lettuce plant of claim 21,wherein the desired trait is herbicide resistance and the resistance isconferred to an herbicide selected from the group consisting ofglyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxyproprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione,triazine, and benzonitrile.
 23. The lettuce plant of claim 21, whereinthe desired trait is insect or pest resistance and the insect or pestresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.